CN1535316A - Transformation method for obtaining marker-free plants and plants obtained therewith - Google Patents

Abstract

The invention relates to a transformation method for obtaining transgenic plants and plants obtained with said method. The invention provides a method for transforming a plant cell comprising providing a plant cell with a recombinant nucleic acid comprising a T-DNA construct allowing for transfer of said construct into the genome of a plant cell, said construct provided with a foreign nucleic acid that is free of nucleic acid encoding a selective marker.

Description

Obtain method for transformation and the thus obtained plant of unmarked plant

Since the last century 70 generation ends and the beginning of the eighties, after Ti (tumor inducing (tumor the inducing)) plasmid of finding edaphic bacillus spp (agrobacteriumspp) can be used as carrier in the genetically engineered plantization, use transform bacteria such as edaphic bacillus spp to transform the method that plant obtains to express interested heterologous gene or gene fragment and know for everybody.Wild plasmid inducing plant cell produces tumour cell, but it can be modified and carries the foreign gene construct and enter cell and need not to make the recipient cell tumourization.In the tumor inducing process, a specific fragment of Ti-plasmids is called T-DNA (transfer DNA (transferred DNA)), is integrated into host plant cell nuclear DNA.In plant genetically engineered, described T-DNA modified and carried can be integrated into DNA of plants the foreign gene construct to obtain transgenic plant.

The step of converting that obtains transgenic plant is normally with a transform bacteria infection plant cell that contains the strain of original non-tumorigenic edaphic bacillus, this bacterium has the T-DNA of containing vector construction body and allows described construct to shift the into genomic recombinant nucleic acid of vegetable cell, described construct comprises the required nucleic acid that hope is expressed basically in final conversion plant, gene or gene fragment and selective marker nucleic acid or selection gene.Heterologous gene that this is required and mark are usually located on one section T-DNA of plasmid or carrier, and this DNA flank has at least one, or are positioned at two of being called as the T-DNA border and are generally between long incomplete (imperfect) forward repetition of 24 base pairs.Shifting heterologous gene/selection gene construct enters vegetable cell and occurs in the transfer and integration process of the regulation and control T-DNA/ gene construct that vir gene (being positioned identical or different plasmid) relates to.Vir-albumen (D1 and D2) produces border multiple breach in accurate site, and the T-DNA construct is cut and insertion Plant Genome from the T-DNA boundary of plasmid.

Vegetable cell after handling is like this grown in suitable environment, and all plants are all renewable, and wherein some have required gene.In order in the cell background of unconverted, to select required transformant, the DNA of the described selection gene of encoding and phraseology thereof usually with the T-DNA that allows the transformant recovery on interested gene being connected physically arranged.It has been generally acknowledged that having such selection or flag sequence on T-DNA is an absolute condition of efficient recovery; Otherwise, thousands of screened to prove existing and functional insertion of required heterologous gene to ten hundreds of supposition transformant needs.Perhaps, the transformant of supposing is always grown under selection substratum or selective pressure, and this is fit to allow selected selective marker make transformant have the selectivity advantage than the non-transformed cell of initial surplus.

In history, plant selectable marker is a dominant gene, can resist the selective substances that adds regeneration culture medium after the expression, but it self acellular growth institute is essential.For example microbiotic, weedicide, amino acid or amino acid analogue add in plant or the plant culture with toxic concentration with such selection material.Most popular selective marker or selection gene in Plant Transformation, the aphIV gene of the bacterium neomycin phosphotransferase gene (nptI, nptII and nptIII gene) of the antagonism of in EP 131623, mentioning selection material kantlex and the antagonism Totomycin of in EP 186425, mentioning.EP 275957 has disclosed the application of the streptomyces viridochromogenes acetyltransferase gene of antiweed L-2-amino-4-(hydroxyl) (methyl) phosphinyl butyric acid (phosphinotricin).In EP 218571, mentioned the plant gene of antagonism herbicide glyphosate (glyphosate).Resistibility is based on coding 5-enol acetone shikimic acid-3-phosphoric acid ester synthase (5-enolshikimate-3-phosphatesynthase, EPSPS) expression of gene relevant with N-phosphoric acid methylglycine (N-phosphomethylglycine) tolerance.Some amino acid is Methionin, Threonine or lysine derivative ethylamine base Gelucystine (AEC) and tryptophane analogue such as 5-methyl tryptophan for example, and growth capable of inhibiting cell when using owing to high density also can be used as the selection material.In this selective system, the expression of selectable marker gene cause can be under selecting the amino acid overproduction of the transgenic cell of growth.

Another kind of selective marker is that those can support the mark that transformed plant cells is grown and bred under the state that non-transformed cell can not fully be grown, and for example, lacks the state of plant growth hormones.Enzyme of a selectable marker gene codified, after it is expressed, to encrypt (encryptic) carbon source is converted to and supports transformed plant cells in the carbon source that is containing growth and breeding under the environment of minimum nutrient, and encrypted carbon source of carbon source or hiding carbon source replace under this environment, can not be utilized by non-transformed cell.Such selective marker for example is a phosphomannose isomerase, it is converted to unavailable seminose-6 phosphoric acid can be by vegetable cell as the fructose-6-phosphate (mentioning among the US 6143562) of carbon source or the xylose isomerase (Haldrup A. etc. 1998, Plant CellReport 18:76-81) of redness of the skin or complexion streptomycete.

Yet another kind of selectable marker gene allows the transformed plant cells of inferring is screened, rather than transformant opposing toxicant such as antibiotic direct heredity are selected.For this reason, they also often are called as screening property mark.These genes are called reporter gene, comprise for example GRD beta-glucuronidase (GUS), β-nougat, luciferase, chloramphenicol acetyltransferase and green fluorescent protein (GFP).

Perhaps, selection markers can provide some other visible active reactions.For example, when material existed, it produced the plant or the relevant unique look or the growth pattern of vegetable cell of expression screening marker gene not, this material or directly apply to plant or vegetable cell be present in plant or the plant cell growth substratum in.

Usually, the plant or the vegetable cell that comprise such screening marker gene have unique phenotype of being convenient to identify, that is, they can differentiate with non-transformed cell.Distinctive phenotype can be identified cell, cell mass, tissue, organ, plant part or the whole plants that comprises this construct.It is the prenyltransferase gene (WO 00/37060 such as Keller) of edaphic bacillus that paramophia is induced the example of (MAI) marker gene.Prenyltransferase is a kind of plant growth hormones---the biosynthetic rate-limiting enzyme of phytokinin.The vegetable cell that has imported the ipt gene produces phytokinin, and the propagation and the differentiation of cell that causes containing the ipt gene is chaotic, and has caused various paramophias.

In the final transgenic plant that obtain, it is undesirable that the existing in of antibiotics resistance gene and other selective marker sequences in most cases thought.Though, think that these sequences are essential to conversion process, transform the useless really of plant for final usually, and in fact owing to a large amount of reason human consumers wants to reduce it.Consumer organization shows concern for the extensive distribution of resistance marker in foodstuff products, proposes to exist transgenosis to select the gene horizontal transfer to enter the theoretical risk of intestinal bacteria.

Environmental organization is paid close attention to the risk of crossing pollination between transgenic plant and the relevant species, and it can cause resistance to be transferred in the weeds, and the life-time service that endangers transgenic crop also causes the potential ecological problem.Therefore, produce unmarked plant and may alleviate the boredom that the public accepts transgenic crop.

So far, develop many systems and promoted to remove selectable marker gene.The cotransformation of two different constructs (co-transformation) can produce the transgenic lines of having integrated two genes and after heredity intersected, selective marker can be kept apart with interested gene.WO 95/16031, and US 6265638 and WO 00/18939 mention by cotransformation and remove selectable marker gene.But cotransformation system requirements marker gene is positioned non-chain position, means the many more independently transformation events of screening.And, many crossing pollinations and particularly vegetative farm crop, especially farm crop tuberous such as potato and cassava are the height heterozygosis, isolate by heredity and remove flag sequence and will need many years just can find clone with suitable wild performance.Several transposable element systems and site-specific recombination system have been used to remove mark, and (Sugita K waits 2000Plant J 22:461-469; Zuo J waits 2001 Nature Biotech 19:157-161).EP 716147 describes a carrier mediated required gene and enters plant, and it comprises interested gene and at least one, and to be placed on resectable DNA element be that paramophia in transposable element or the site-specific recombination system is induced (MAI) marker gene.The plant that conversion contains gene construct MAI can be easy to by the abnormal morphology of visual inspection branch detect.Similarly, behind the transposable element swivel base or after the recombination system site-specific excision, the branch of normal morphology occurs, be easy to detect losing of MAI gene function by observation.Need not intersect step, can generate the transgenic plant of such marker-free.These system requirements are expressed a transposase or recombinase, and it mediates the disappearance of the equal area between reorganization or the transposase target sequence and isolates by heredity subsequently and remove marker gene.And for being mainly vegetative species, these systems are time-consuming, also are unpractical.And the fragment of disappearance can be inserted genomic other positions again.The method of another inducing DNA disappearance is a homologous recombination in the karyomit(e) that makes up between 2 homologous sequences.The homologous recombination frequency is extremely low for the disappearance that this system of effective application produces the transgenosis zone in the karyomit(e).Utilize the attP district of phage can improve this recombination frequency (2000 Nature Biotech 18:442-445 such as Zubko E.).But this process is still unpredictable and efficient is too low so that can not produce hundreds of clones that find to be suitable for agriculture production to thousands of independently transformation events.The system that does not use selective marker to reclaim transgenic cell is described.In WO 98/51806, disclosed a method of not using the recovery transformant of selective marker by using joint culture (nodal culture) and non-selective screening to analyze enrichment transgenosis part.This method relate to comprise nonselective analyzable genetically modified cultivation transformed plant cells or the tissue comprise merismatic node up to having grown.Subsequently, use a non-selective determination and analysis plant tissue, as enzyme analysis or ELISA.The analysis sun plant of reclaiming with this method is chimeric and transform portion is arranged.From these transform portion of explant enrichment of positive knot of tissue and cultivate into branch.Re-use non-selective determination and analysis branch and leaf, wish to have reclaimed the plant of being rich in transform portion so that finally through several take turns analysis after, obtain transgenic plant near homogeneous.

Summary of the invention

The invention provides the method that produces transgenic plant, described transgenic plant are crossing pollination and vegetative farm crop particularly, it comprises a required gene, and therefore auxiliary nucleic acid of essentially no other external sources or allos such as selectable marker gene do not need through selecting mass treatment to screen.The system of all above-mentioned unmarked transformants of mentioning of generation all supposes to separate the non-chimeric transformant that does not contain selective marker and is actually infeasible, and it is not all right using the conversion scheme of T-DNA construct or corresponding transform bacteria at least.Can produce the plant that comprises required gene in this method that provides, but it is not used in carrier sequence and/or the flag sequence that transforms plant basically.Surprisingly, the invention provides the method that transforms plant or vegetable cell, comprise and use transform bacteria such as edaphic bacillus spp, obtain to express the non-chimeric transgenic plant of required interested heterologous gene with its T-DNA, wherein said T-DNA has required gene or gene fragment, but does not comprise extra selection gene or its fragment.

For this reason, the invention provides the separation or reorganization nucleic acid that contains T-DNA or its functional equivalents, it allows described T-DNA to shift the into genome or the nuclear DNA of vegetable cell, described T-DNA has the nucleic acid of the nucleic acid that does not contain the selective marker of encoding, and the such application of T-DNA construct in genetically engineered plantization is provided.Therefore, method provided herein need or not be in the transformant of growth supposition under the selection pressure in selective medium and real conversion plant is occurred.On the contrary, the selection of required transformant is easy to realize by the existence of checking required heterologous gene or gene fragment or self construct, for example, use the nucleic acid technology of knowing, as the complementary sequence hybridization method of polymerase chain reaction detection or conventional Southern Blot experiment.Obviously to those of skill in the art, the existence by the gene expression product or the change of disappearance or amount can be analyzed the existence of required heterologous gene or gene fragment.For example, the expressing protein that an available ELISA (enzyme-linked immunosorbent assay) detects is with this proteic existence of elisa assay.And method provided herein can relate to biological assay or chemical analysis method such as gas chromatography/mass spectrum (GC/MS).Need shift the reorganization T-DNA construct that described T-DNA construct enters plant cell dna to being used to, construct existence or the disappearance partly that particularly has the exogenous nucleic acid of required nothing coding selective marker nucleic acid detects.The preferred nucleic acid of the present invention comprises a T-DNA construct, and it comprises at least one T-DNA border, but integrates in order to be easy to, and there are T-DNA border tumor-necrosis factor glycoproteins or its functional equivalents in the both sides of preferred described exogenous nucleic acid.The VirDl of edaphic bacillus host cell and VirD2 albumen identification border tumor-necrosis factor glycoproteins, and between third and fourth base of each border repetition chain end, produce a strand otch.These otch determined to lay respectively at border, the left and right sides the T chain open stop bit point all the time.Find that left margin is nonessential for the transfer of T-DNA, but it helps definition to define the left-end point of T-DNA.As if right margin shift of crucial importance for T-DNA.The Ti-plasmids of T-DNA right margin zone disappearance does not have virulence (Holsters, Plasmid such as M. 3,212-230,1980) usually.Disappearance left margin zone does not have influence for virulence.The sequence context of tumor-necrosis factor glycoproteins has determined their relative reactivities when T-DNA shifts.(Mol.Gen.Genet.210 such as Wang, 338-346,1980).The border multiple example of a typical pBIN19 plasmid is right margin 5 '-TGACAGGATATATTGGCGGGTAAAC-3 ' and left margin 5 '-TGGCAGGATATATTGTGGTGTAAAC-3 '.Certainly, be easy to realize the present invention with nucleic acid of the present invention, wherein said Ti-DNA derives from the Ti-plasmids of edaphic bacillus spp, and particularly wherein said edaphic bacillus comprises Agrobacterium tumefaciens (A.tumefaciens), and described bacterium and technical skill can extensively obtain.

In one embodiment, provide nucleic acid of the present invention, wherein said exogenous nucleic acid can be regulated and control target gene expression in the described genome.Can realize raising (or cross and express) and downward modulation by " sense " technology.If the target gene of total length copy has inserted genome, then can obtain a series of phenotypes, some cross the expression target gene, some low expression.Screen the plant population of producing with this method and separate each phenotype.A preferred embodiment comprises a method, and wherein said regulation and control comprise downward modulation.Suppress a target gene expression, be commonly referred to " gene silencing ", can realize by " antisense downward modulation " and " the justice downward modulation is arranged " (being also referred to as " suppressing (cosuppression) altogether ").In antisense downward modulation, with all or part of complementary dna fragmentation of endogenous target gene to insert genome in the other direction.Though also do not illustrate mechanism fully, a theory is that such inverted defined gene is transcribed the mRNA and the complementation on sequence of endogenous gene mRNA transcription product of generation.Antisense mRNA combines natural mRNA of inhibition of formation and translates into proteic duplex with " justice is arranged " mRNA of natural generation.Conditioning technology is known for those skilled in the art and conventional use the in global laboratory under the antisense.Therefore, the segmental copy of at least one of target gene encoding sequence is inserted the genome of target organism and realize gene silencing, its copy comprises or the sequence of all or part of or brachymemma and for justice or antisense orientation are arranged.In addition, use may be obtained from the intron sequences structure inhibition carrier of genomic gene sequence.Report is arranged, in organism, realized the gene silencing of transgenosis and endogenous gene, wherein sequence unanimity in promoter region only.

In a preferred embodiment, the invention provides a T-DNA construct, wherein said exogenous nucleic acid comprises the reverse repetition in the polynucleotide zone of the described target gene of at least one part.Though, antisense and have adopted downward modulation can cause target gene silence completely, efficient is not very high usually.There have 25% antisense transformant to present at most to be reticent completely, and the transformant that 10% the usefulness of only having an appointment has adopted construct to obtain shows silence (Smith etc., 2000, the Nature 407:319-320 of certain level; Wolters and Visser, 2000, Plant Mol Biol 43:377-386).Recently, observing when the gene silencing carrier comprises that all or part of the polymerized nucleoside acid region of a target gene oppositely repeats, is enhanced to selecting the inhibition of target gene in the organism.Inverted repeats is made up of a T-DNA, it has a manipulation an adopted cDNA copy expression promoter and another promotor (Chuang and a Meyerowitz who is positioned at the antisense copy front of identical cDNA, 2000 Proc Natl Acad Sci USA 97:4985-4990), perhaps this T-DNA is included in the cDNA sequence (2000 Mol Biochem Parasit111:67-76 such as LaCount) that there is promotor in 2 terminal flanks, and perhaps this T-DNA comprises the promotor that the inverted repeats of handling this cDNA (part) transcribes (Hamilton etc. 1998; 2000 Nature 407:319-320 such as Smith; Wang and Waterhouse, 2000 Wang MB, Waterhouse PM (2000) Plant Mol Biol43:67-82), perhaps by the promotor of silencer (2000 EMBO J 19:5194-5201 such as Mette).It is reported that when using an intron some oppositely repeat construct and have caused 100% transformant to present target gene silence 2000Nature 407:319-320 such as () Smith as the transcribed spacer between tumor-necrosis factor glycoproteins.Stuffer fragment has increased the stability of correct inverted repeats, but optional for the specificity of silence.In a particular preferred embodiment of nucleic acid of the present invention, and described target gene coding starch small grain constraint starch synthase (granule-bound starch synthase, GBSSI).The reticent fully transformant occurrence frequency of herein describing in detail at the target gene of coding transgenic Rhizoma Solani tuber osi starch small grain constraint starch synthase (GBSSI) in back, the multiple zone of short weight has remarkable increase.

Nucleic acid of the present invention also is provided, wherein said exogenous nucleic acid can be in described vegetable cell the expressing heterologous polypeptide.Suitable polypeptide is multi-form, and required conversion non-marked enters the exogenous nucleic acid of plant or the representative instance of gene is albumen and the enzyme that those codings are modified metabolism and catabolic process.Other examples are proteic genes of codified nutritive value when increasing plant as food or farm crop.Typical example comprise can suppress the vegetable-protein that antinutritional factor forms and have how amino acid needed composition (as, have higher lysine content than non-transgenic plant) vegetable-protein.One preferred embodiment in, described nucleic acid or required genes encoding comprise the polypeptide of enzyme.The enzyme that required gene codified uses in the foodstuff processing process is as rennin, strange (different fruit) monellin and α-nougat.The material that required gene also can be encoded and be introduced or increase pathogen resistance.Required gene codified helps the non-natural plant mixture of animal or human's class.For example, required gene codified medicine activated protein or enzyme such as any one treatment compound such as Regular Insulin, Interferon, rabbit, human serum albumin, human growth factor and thrombin.In this, transformant or organism can prepare desirable very easy in the desired mixt as reclaiming the stem tuber.Preferably, required gene is albumen or peptide, the insensitive amino acid biosynthetic enzymes class of feedback such as the dihydro-2 that coding has high nutritive value, dipicolimic acid 2 synthase (dihydrodipicolinate synthase) (EC 4.2.1.52, DHPS), have disease resistance enzyme or peptide, have justice or antisense to transcribe gene as potato tuber storage protein, ADP-glucose pyrophosphorylase, α-Dian Fenmei, q enzyme, starch small grain constraint starch synthase, soluble starch synthase (sss), proteolytic enzyme or dextranase.

The present invention also provides carrier or plasmid that comprises nucleic acid of the present invention and the host cell that comprises such carrier or nucleic acid.A preferred host cell comprises edaphic bacillus.To operating method for transformation of the present invention, preferably use abundant fatefulue edaphic bacillus such as Agrobacterium tumefaciens, obtainable other toxicity strains in the bacterial strain in for example representational A281 strain or its source or this area.These edaphic bacillus strains have a DNA zone that derives from the virulence zone of the Ti-plasmids pTiBo542 that comprises virB, virC and virG gene.The processing that the virulence gene of Agrobacterium tumefaciens (vir gene) product is coordinated T-DNA enters the conversion of vegetable cell with it.Vir genetic expression is by virA and virG control, and wherein by phosphorylation, virA activates virG after experiencing inducement signal.VirG induces the expression of virB, C, D, E successively.The albumen of these genes encodings relates to the conversion of DNA.Mol.Gen.Genet 230:302-309 such as (, 1991) Chen that the PTiBo542 virulence strengthens that the super toxicity virG gene be considered on the Ti-plasmids causes.But, except that usability metachromia edaphic bacillus, when people wish to implement when of the present invention, exist other method can effectively transmit DNA and enter the recipient plant cell.Transmit any method that appropriate method that DNA enters vegetable cell in fact comprises the DNA transfered cell, direct transmission (Omirulleh etc. as the DNA of the conversion protoplastis of PEG mediation, 1993), DNA dry or that suppress to mediate absorbs (Potrykus etc., Mol.Gen.Genet., 199:183-188,1985), electroporation (the U.S. patent No. 5,384,253), the vibration of silicon carbide optical fiber (Kaeppler etc., 1990; U.S. the patent No. 5,302, and 523; With the U.S. patent No. 5,464,765), DNA wraps by particulate acceleration method (the U.S. patent No. 5,550,318; U.S. the patent No. 5,538, and 877; With the U.S. patent No. 5,538,880).By using these technology, any floristic some cell can transform with being stabilized, and these cell developments are transgenic plant.

In little bullet method, particle can enter cell by nucleic acid bag quilt and through the impellent transmission.Representational particle comprises the particle that those are made up of tungsten, gold, platinum and resemblance.Expection in some cases, DNA is deposited on the metallic particles for using little bullet method that DNA is passed to recipient cell optional.But the expection particle may comprise DNA rather than be wrapped quilt by DNA.With the corresponding to little bullet conversion method of present invention, physics and biotic factor all can be optimised.Physical factor is that those relate to operation DNA/ particulate deposits thing or those influences big or the range of molecule and the factor of speed.Biotic factor comprise relate to before the bombardment and the institute of afterwards manipulated cell in steps, regulate and control to help to alleviate the relevant damage of bombardment as the perviousness of target cell, the orientation of the target tissue relevant with the particle trajectory and the character of transfering DNA are as linearizing DNA or complete supercoiled plasmid.It is believed that the operation before the bombardment is very important for successfully transforming.

Therefore, people wish studying the different bombardment parameter of adjustment among a small circle to obtain complete optimized conditions.It may be DNA concentration, clearance distance, flying distance, tissue distance and helium pressure that people wish to adjust physical factor especially.The rank that further contemplates that helium can influence transformation efficiency.People also can optimize damage minimizing factor by changing the environment that influences the recipient cell physiological status and therefore influence conversion and integration efficiency.For example, adjust the infiltration state, organize the cell cycle of hydration and inferior incubation period or recipient cell to transform to optimize.The transfering DNA that is used for particle conversion comprises an expression cassette, and it contains the cDNA that is hopeful transfered cell usually, a gene or several gene, and and then comprise handling and connect exogenous gene promoter and 3 ' zone.Optimized gene construct has been described in the present invention.Dna fragmentation additionally comprises the the the 2nd, the 3rd, the 4th, the 5th, the 6th or any being placed on the single DNA molecules and being transformed the foreign gene that enters recipient cell of additional number.

In the specific embodiments of the present invention, dna fragmentation does not comprise transforming to be selected or the selection markers gene.Usually, except that required gene, people use selection or selection markers gene to improve the ability of differentiating transformant." marker gene " is to give the gene of the clear phenotype of cell of presentation markup gene, therefore can distinguish such transformant and unmarked cell.Such gene can be encoded and be selected or selection markers, depend on that mark whether given the characteristic that can select by chemical mode, promptly, by using selective substances (as weedicide, microbiotic, amino acid analogue or resemblance), perhaps whether it only is a characteristic of differentiating by observation or test, that is, by " screening " (as R-locus (R-locus) characteristic, β-Pu Taotanggansuanmei or uidA gene).Transformant for production carrier free dna sequence dna, hope transmission DNA enters and does not comprise the cell of keeping the necessary dna sequence dna of plasmid vector in the host bacterium, as E.Coli, antibiotics resistance gene for example, include, but are not limited to ampicillin, kantlex and tetracyclin resistance, and the protokaryon starting point of dna replication dna.In this case, the dna fragmentation that comprises transfering DNA can be purified before conversion.For example on sepharose, carry out gel electrophoresis and realize purifying, reclaim dna fragmentation from sepharose subsequently.

The transgenosis number of copies that the modification may command of the nucleic acid fragment end being carried out through flat endization of dephosphorylation or nucleic acid fragment is introduced and effectively production hang down and copy transformant (WO 99/32642).The present invention may derive from any transformable unifacial leaf of potential or dicotyledons with the recipient plant cell that little bullet method transforms.The preferred monocot plant cell that the present invention uses derives from rice, wheat, barley, oat, rye, grain, Chinese sorghum, sugarcane, lawn and corn.The preferred dicotyledons cell that the present invention uses comprises cotton, tomato, oranges and tangerines, tobacco, soybean and special potato and cassava.

After realization entered recipient cell with the foreign DNA transmission, next step related to identification of transformed cell so that further cultivate and aftergrowth usually.In the present invention, having transmitted the cell behind the foreign DNA cultivates in the substratum of supporting plant regeneration.After branch is grown, use DNA or RNA detection method to detect and comprise one or more genetically modified seedlings.Method comprises establishing criteria method isolating nucleic acid from seedling or one group of seedling.(Sambrook etc., 1989).The invention provides use the nucleic acid detection method decision whether a transformed plant cells or its filial generation transformed recombinant nucleic acid, this method comprises the gene product whether described cell of test or described filial generation exist or lack described nucleic acid or its source.Nucleic acid can be genomic dna, RNA or mRNA.For example, use such method to test out the existence and the rearrangement of the special gene in the present gene construct, that is, whether a vegetable cell is fully transformed to use a kind of quality control checking.The invention provides and use the mRNA detection method to determine the nucleic acid construct of plant transformed cell whether or its filial generation fully to be integrated into Plant Genome and to be transcribed into the mRNA construct.Use direct (DNA) or indirect (RNA) amplification method in sample, to identify the genetically modified required specific nucleic acid of part.Secondly, detect the product of having identified.In some applications, detect (as, the ethidium bromide staining of gel) by visual method.Perhaps, detect relate to chemoluminescence, radio-labeled or fluorescently-labeled radioactivity scitiphotograph or even electricity or thermal pulse signalling system identify product indirectly.The transgenic plant of using a series of different testing method screenings to use method of the present invention to produce.These technology are used to detect the existence of special gene and may appear at rearrangement in the gene construct.These technology include, but are not limited to, polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), directly dna sequencing, PFGE analysis, Southern or Northern trace, single stranded conformational analysis (SSCA), the analysis of RNA enzyme protection, allele specific oligonucleotide oligonucleotide (ASO), Dot blot analysis, denaturing gradient gel electrophoresis, restriction fragment length polymorphism (RFLP) and PCR-SSCP or based on the chip of dna technique.The invention provides the application nucleic acid detection method and determine whether that plant transformed cell or its filial generation are transformed by recombinant nucleic acid, this nucleic acid comprises one and is integrated into genomic T-DNA construct of vegetable cell or functional nucleic acid construct of equal value.And, the invention provides use the decision of nucleic acid testing method whether a transformed plant cells or its filial generation transformed by recombinant nucleic acid, the non-required solid support material that comprises described cell of test or described filial generation is the existence or the disappearance of carrier main chain sequence for example.For example, use method of the present invention to detect whether the plant transformed cell does not have auxiliary non-required nucleic acid basically.Differentiate the seedling that transforms with the nucleic acid testing method, allow it develop into plant then.Aftergrowth enters stem and after the root development stage, they can be moved into greenhouse further growth and test.

In addition, the invention provides vegetable cell that comprises nucleic acid of the present invention and the aftergrowth that derives from such vegetable cell, or its part.Provide tuberous plant or its part especially, it preferably is selected from potato or cassava plant population.Such potato provided herein or cassava plant comprise so-called unmarked high amylopectin starch stem tuber (for example potato or cassava), because the coding starch small grain fetters the downward modulation of starch synthase gene and lacks amylose starch basically.The amylose starch capacity that such potato or cassava plant contain is less than 5%, and is substantially devoid of any conversion selectable marker gene for example microbiotic or anti-herbicide gene.In addition, in the genome of preferred these plants, there not be to integrate the carrier sequence that is used for conversion process, for example all or part of edaphic bacillus Ti-plasmids and comprise the T-DNA insertion of low copy number, a preferred copy.High-lysine plant (preferred potato or cassava) also is provided, its Methionin that contains surpasses 20% of total free amino acid content, or methionine(Met) surpasses 15% of total free amino acid content, or surpass 30% amylose starch, or surpass 2% solidifying protein, do not transform selectable marker gene for example microbiotic or anti-herbicide gene and do not comprise basically, in their genome, there is not to integrate the carrier sequence that is used for conversion process, for example all or part of edaphic bacillus Ti-plasmids, and the T-DNA that comprises low copy number inserts a preferred copy.

The invention provides the method for transformed plant cells, comprising provides recombinant nucleic acid of the present invention to vegetable cell, comprise the described cell of test or to the existence or the disappearance of the gene product in nucleic acid described in the described cell filial generation of small part or its source, wherein said test comprises the use of nucleic acid detection method.In one embodiment, use the transformant population of nucleic acid detection method screening supposition to detect the existence or the disappearance of exogenous nucleic acid in the vegetable cell in its filial generation source, under the background of no transformed cells, differentiate required transformant.Especially, the nucleic acid testing method, PCR method for example is used to differentiate the tuberous plant cell of conversion, it preferably is selected from potato or cassava plant.In one embodiment, the invention provides the generation that do not comprise selectable marker gene and show that with separating native gene silence or native gene cross the method for the transformant of expression.

At this, we show this method of use even may obtain the transformant of essentially no marker gene, carrier free dna backbone to have only the insertion of 1 T-DNA, the wherein reticent or expression excessively of native gene.In addition, verified this method can produce homogeneous and non-chimeric transformant.In another embodiment, provide the production and the separation method of the transformant of expression of heterologous genes, similarly, essentially no mark of described transformant and carrier DNA main chain.Can further cultivate and obtain to have the unmarked basically plant of required gene with the transformant of method discriminating of the present invention.The invention provides use the decision of protein testing method whether the recombinant nucleic acid construct in transformed plant cells or its filial generation be integrated into Plant Genome fully and regulate and control the genomic expression of target gene of vegetable cell.Protein testing method of the present invention also can be used for determining the expression level of vegetable cell internal protein, and wherein genetic expression is regulated and control, and for example, adopts gene silencing or following conditioning technology to suppress expression of target gene.Adopt enzyme analysis or immunoassay, for example, the expression level of crossing that the downward modulation degree of target gene after the exogenous nucleic acid transformed plant cells or gene are determined in elisa assay, Western trace, Dot blot analysis or similar experiment carries out the selection of transformant.Protein testing method of the present invention can check in certain etap of vegetable cell or its filial generation whether express exogenous nucleic acid in vegetable cell or plant tissue.Such exogenous nucleic acid codified heterologous polypeptide, for example an enzyme.As state, a method is provided, wherein need under the selective pressure of selecting substratum, not cultivate real transformant to get rid of non-transformed cell, use the transformation efficiency of the inventive method very high so that no longer need to select to cultivate.The invention provides the method for transformed plant cells, it comprises provides the recombinant nucleic acid that contains T-DNA construct or its function equivalent to vegetable cell, wherein said vegetable cell is the tuberous plant cell, comprise the described tuberous plant cell of test or the described nucleic acid in its parton generation or the existence or the disappearance of its gene product at least, wherein said test comprises uses the nucleic acid testing method.A method of the inventive method is suitable especially to obtain unmarked tuberous plant, because tuberous plant is the height heterozygosis and passes through conventional heredity and separate not outbreeding outcross marker gene basically.In the present invention, require to test the existence or the disappearance of the functional at least part of nucleic acid of the present invention to the filial generation of the described cell of small part.Such filial generation is by part root or branch, or the individual cells composition, therefore, and for further regeneration and vegetative propagation stay competent converting material.Preferred by using nucleic acid testing method and/or phenotypic screen to select the branch of transformant as transforming.Obtainable testing method detects the transformant that has required gene and do not have auxiliary nucleic acid in employing Southern hybridization, pcr program and other this areas.Such test can further comprise to test and exist to the filial generation of the described cell of small part or lack non-required vector nucleic acid.In brief, this invention provides the method for producing the transgenic plant that comprise required gene and essentially no auxiliary non-required nucleic acid.In a preferred embodiment, the invention provides the optimized step of converting that transforms plant, use (preferably having virulence) edaphic bacillus strain, the effective ways of the gene construct of the effective inhibition optimized or mistake expressing gene and biochemical test rather than selective pressure test transformant, for example, by Southern blot hybridization, polymerase chain reaction (PCR) program or other obtainable nucleic acid testing method.

Another preferred embodiment in, the invention provides the method that transforms the tuberous plant cell, the recombinant nucleic acid that the T-DNA construct that contains the nucleic acid that has or not the selective marker of encoding is provided is to described cell, described construct has the target gene of coding starch small grain constraint starch synthase (GBBI), comprise the existence or the disappearance of testing to target gene described in the filial generation of the described tuberous plant cell of small part, wherein said test comprises the use nucleic acid detection method, for example uses round pcr.

Description of drawings

Fig. 1 .GBSS cDNA inverted defined gene silent carrier collection of illustrative plates

Fig. 2. plasmid pMTL1.1 collection of illustrative plates, this plasmid comprise 5 ' end of 1.1 kb of the GBSS cDNA that is used to prepare Fig. 4,5,6,7,8,13,14 and 15 plasmids

Fig. 3. plasmid pMTL1.3 collection of illustrative plates, this plasmid comprise 3 ' end of the 1.3kb of the GBSS cDNA that is used to prepare Fig. 4,5,6,7,8,13,14 and 15 plasmids.

Fig. 4. have the GBSSI gene silencing carrier pKGBA50-IR1.3 of nptII selectable marker gene, it comprises by 3 ' end of the 1.3kb of the cDNA of the GBSS of the terminal reverse repeating pattern that interrupts of 5 of the 1.1kb of GBSS cDNA '.

Fig. 5. have the GBSSI gene silencing carrier pKGBA50-DR1.3 of nptII selectable marker gene, it comprises by 3 ' end of the 1.3kb of the GBSS cDNA of the terminal series connection repeating pattern that interrupts of 5 of the 1.1kb of GBSS cDNA '.

Fig. 6. have the GBSSI gene silencing carrier pKGBA50-IR1.1 of nptII selectable marker gene, it comprises by 5 ' end of the 1.1kb of the GBSS cDNA of the terminal reverse repeating pattern that interrupts of 3 of the 1.3kb of GBSS cDNA '.

Fig. 7. have the GBSSI gene silencing carrier pKGBA50-DR1.1 of nptII selectable marker gene, it comprises by 5 ' end of the 1.1kb of the GBSS cDNA of the terminal series connection repeating pattern that interrupts of 3 of the 1.3kb of GBSS cDNA '.

Fig. 8. the GBSSI gene silencing carrier pKGBA50mf-IR1.1 of marker-free, it comprises by 5 ' end of the 1.1kb of the GBSS cDNA of the terminal reverse repeating pattern that interrupts of 3 of the 1.3kb of GBSS cDNA '.

Fig. 9. have the conversion results of the potato plants cv.Karnico of 4 different GBSSI gene silencing constructs, compare with traditional GBSSI antisense constructs, these constructs comprise GBSSI cDNA reverse or the series connection repeating pattern.After picking out transformant, to the plant adding stem tuber inducing culture of growth in vitro.After 2 to 6 weeks, most branches grow the test tube potato.Downcut the starch iodine staining of stem tuber surface gained.Contain (nothing) of being dyed the particulate transformant right and wrong silence of blue look by iodine solution, the stem tuber of red coloration be reticent (fully/consumingly) and the transformant of " weak or medium " color indicating section silence.

Figure 10. comprise the collection of illustrative plates of the plasmid pAAP105mf of the potato DHDPS cDNA that is subjected to the control of GBSSI promotor that feeds back insensitive no Plant Transformation selective marker.

Figure 11. the free lysine concentration (accounting for the % of total free aminoacids total amount) that derives from the test tube potato of different independent transformants or control plant is induced stem tuber to the plant adding stem tuber inducing culture of growth in vitro.After 2 to 6 weeks, grow the test tube potato for most of.To organize (0.5-1.0 gram) to contain homogenate in the Pi damping fluid of 1mM dithiothreitol (DTT) at 2ml.Add nor-leucine as inherent standard.Use 5ml water: chloroform: carbinol mixture (3: 5: 12) extracts partial purification free amino acid.After freeze-drying is concentrated to 3ml, gets 20 microlitre samples and analyze with HPLC cationic exchange coloum (BIOCHROM 20, Amersham Pharmacia biotech).

Figure 12. plasmid pAAP172 collection of illustrative plates, it comprises the insensitive potato DHPS*cDNA that is subjected to the control of potato GBSSI promotor that is used to prepare Figure 13,14 and 15 plasmids of feedback of 1.1kb.

Figure 13. make up GBSSI gene silencing carrier pKGBA50mf-R1.1, it comprises the insensitive potato DHPS*cDNA of feedback of pAAP105.

The T-DNA of Figure 14 .pDHPS-IR1.1 (series connection), its comprise GBSS cDNA 1.3kb the 3 ' terminal reverse repeating pattern that interrupts GBSS cDNA 1.1kb 5 ' end and do not have the insensitive potato DHPS*cDNA of feedback of the series connection direction of Plant Transformation selective marker.

The T-DNA of Figure 15 .pDHPS-IR1.1 (series connection), its comprise GBSS cDNA 1.3kb the 3 ' terminal reverse repeating pattern that interrupts GBSS cDNA 1.1kb 5 ' end and do not have the insensitive potato DHPS*cDNA of feedback of the inverse direction of Plant Transformation selective marker.

That Figure 16 .Southern engram analysis separates is unmarked, carrier free and do not have the DNA of the Transformation of potato strain of amylose starch.Isolation of genomic DNA from 17 transformants and a unconverted contrast strain (Karnico), use HindIII digests and seeks and visits with NOS terminator probe.

Embodiment

The optimization of Plant Transformation

The high-throughput that the present invention mentions is produced unmarked transformant and is required not rely on genotypic efficient transformation frequency.Optimization conversion scheme single and dicotyledons is the problem of many researchs.For transforming tuberous plant such as potato, some method for transformation are delivered.In many methods of these schemes, transformation efficiency is relied on genotype very much.Whether the difference of essence exists plant hormone or there is not growth hormone in callus after the initial stage and has Plant hormones regulators,gibberellins between different schemes in the whole regeneration period.That nutrient media components in the scheme of delivering and growth hormone and cytokine type and level also exist is different (1992 Plant Molec.Biology 31 such as Hulme, 161-167).But a large amount of investigation shows ethene and piles up the regeneration of inhibition of potato plant, this restraining effect when having ethene biosynthesizing or ethene activity inhibitor, be overcome (1988 Plant Cell Rep.7 such as Perl, 403-406).Therefore, some schemes add silver thiosulfate (STS) in regeneration culture medium.The conversion scheme can be carried out other and be revised to increase transformation efficiency.For example, after WO 0034491 mentions the inoculation edaphic bacillus, reduce wet condition and make explant weight saving can improve transformation efficiency.Comprise that in the possible method that alleviates explant weight altogether between incubation period the osmotic pressure, the interpolation siccative that increase substratum comprise calcium oxide or sulfuric acid or air-dry explant.

But for gyp conversion, single is easily for the effective scheme of Cultivar on a large scale.Than relatively large different scheme about a series of potato cultivars of regenerating, (1992 Plant Molec.Biology 31 such as Hulme, 161-167) reach a conclusion, compare with those so far published a large amount of potato cultivar schemes, (1988Euphytica 39, and scheme 213-219) is better for Hovenkamp Hermelink etc.At this, we prove that 2 transform scheme about regenerated the quantity of the unmarked transformant that obtained is had material impact.

The optimization of transformation efficiency

The edaphic bacillus strain that preferably is used for the inventive method is modified comprises required gene, or the nucleic acid of expressing in transformant.The nucleic acid that is transferred is mixed the T zone and there is at least one T-DNA border sequence in flank.Multiple edaphic bacillus kind known in the art to transforming dicotyledons, comprises Agrobacterium tumefaciens and rhizobiaceae especially.See, for example, Hooykaas, P.J.1989Plant Mol.Siol.13:327; 1995 Crop Science 35:301 such as Smith; Chilton, M.O.1993 Proc.Natl.Acad.Sci.USA 90:3119; 19:148; 1996 NatureBiotechnol.14:745 such as Ishida; Komari, 1996 The Plant Journal 10:165 such as T.In Ti-plasmids, the T zone is different with the vir district that is responsible for transfer and integration function.The binary vector system is developed, and steerable T-DNA and the vir function that has foreign DNA is present on the isolating plasmid.Like this, comprise foreign DNA (the required nucleic acid that is transferred) modification the T-DNA zone be structured in the miniplasmids that duplicates among the E.Coli.Adopt triparental mating or electroporation or freeze molten program this plasmid is engaged in the Agrobacterium tumefaciens of the compatible plasmid that transforms to advance to comprise to have virulence gene.Transfer T-DNA enters Plant Genome in trans provides Vir function.The existence in severe toxicity vir zone has a significant impact transformation efficiency.The preferably Agrobacterium tumefaciens strain of the double base of agropine.Agrobacterium tumefaciens strain EHA101, AGL0 or AGL1 that especially preferably can public acquisition.These bacterial strains comprise the C58 bacterial chromosome and result from the disarmed derivative of the Ti-plasmids that is called TiBO542 in the literature of hypertoxic Agrobacterium tumefaciens A281.[see, for example, Hood E.E. etc., (1986) J.Bacteriology 168:1291-1301, (1991) Biotechnology9:963-967 such as Lazo GR.].

Perhaps, the edaphic bacillus strain that is used comprises to have and strengthens toxic plasmid.Preferably put into practice the present invention with super binary vector.Made up so super binary vector, it has comprised DNA zone (Hood etc. (1984) Biotechnol.2:702-709 in Ti-plasmids pTiBo542 (Jin etc. (1987)) the virulence zone in the Agrobacterium tumefaciens A281 that demonstrates high transformation efficiency; Hood etc. (1986) J.Bacterial.168:1283-1290; Komari etc. (1986) J.Bacteriol.166:88-94; Jin etc. (1987) J.Bacterial.169:4417-4425; Komari T. (1989) Plant Science 60:223-229; ATCC Accession No.37394).

Super binary vector comprises pTOK162 (Japanese patent application No. (Kokai) 4222527, EP-A-504,869, EP-A-604,662 and U.S. Patent number 5,591,616) and pTOK233 (Komari, T. (1990) Plant Cell Reports 9:303-306; With (1996) NatureBiotechnology 14:745 such as Ishida).Super binary vector pTOK162 duplicates in E.coli and Agrobacterium tumefaciens.In addition, carrier comprises virB, virC and the virG gene in pTiBo542 toxicity zone.Through triple cross, plasmid is imported into edaphic bacillus strain (Ditta G etc., 1980; Proc.Natl.Acad.Sci.USA 77:7347-7351).Except that the edaphic bacillus plant type, known interpolation phenolic compound such as Syringylethanone can strengthen transformation efficiency (seeing Townsend, J.A. etc., US5,563,055) to the edaphic bacillus suspension.The typical concn scope is in 100-200 μ M scope.

The optimization of gene construct

Suppress target gene expression, be commonly referred to as " silence ", can realize by " antisense downward modulation " and " the justice downward modulation is arranged " (being also referred to as " suppressing altogether ").In the antisense downward modulation, insert genome with opposite direction with all or part of endogenous target gene complementary dna fragmentation.This mechanism is not illustrated as yet fully, and theory is that the mRNA that transcribes generation of this inverted defined gene and mRNA that native gene is transcribed are complementary on sequence.So the adopted mRNA that has of antisense mRNA and natural generation suppresses natural mRNA and is translated as protein in conjunction with forming dimer.

Conditioning technology is known in the art and is often used in laboratory, the whole world under the antisense.Cross and express and reduce and all can realize by " justice is arranged " technology.If the total length of target gene copy inserts genome then can obtain a series of phenotypes, the expression target gene excessively that has, the low expression that has.The plant that present method produces can be screened with separate individual phenotype.

Therefore the gene silencing genome that can insert target organism by the additional copy with target gene is realized, the sequence of this copy coding can comprise all or part of or the sequence of having blocked can be that justice or antisense orientation are arranged also.In addition, may can be used to from the intron sequences that the genomic gene sequence obtains make up suppress carrier.Had to be reported in the gene silencing of having realized transgenosis and native gene in the reincarnation object, wherein only had sequence identity at promoter region.

Although antisense and have adopted downward modulation can cause the complete silence of target gene, efficient is not very high usually.Antisense transformant maximum 25% occurs reticent fully, and has only about 10% silence (the 2000 Nature 407:319-320 such as Smith that certain level occurred by the transformant that has adopted construct to obtain; Wolters and Visser, 2000 Plant Mol Biol 43:377-386).Recently, observed when the gene silencing carrier comprises all or part inverted repeats of polynucleotide of target gene, target gene selected in the organism suppresses to have improved (mentioning as WO 99/61632, WO98/53083 and WO 99/53050).

It can be to have T-DNA or this T-DNA of another promotor (Chuang and Meyerowitz, 2000 Proc Natl Acad Sci USA 97:4985-4990) that drives cDNA and justice copy expression promoter is arranged and be positioned at the antisense copy front of identical cDNA to comprise cDNA sequence (the 2000 MolBiochem Parasits 111 such as LaCount of two ends by the promotor linking that inverted repeats is formed; 67-76), or this T-DNA comprise and drive the promotor that cDNA (part) inverted repeats transcribes (Hamilton etc. 1998; 2000 Nature 407:319-320 such as Smith; Wang and Waterhouse, 2000 Wang MB, Waterhouse PM (2000) Plant Mol Biol 43:67-82) or the promotor (2000 EMBO J 19:5194-5201 such as Mette) of gene by reticent.

When using intron as the tumor-necrosis factor glycoproteins interval, some oppositely repeats the silence (2000 Nature 407:319-320 such as Smith) that construct is in the news and has caused 100% transformant display target gene.Stuffer fragment has been given the stability of correct inverted repeats, but optional to the specificity of silence.

We have proved and have surpassed potato that 60% GBSSI transforms and wild-type relatively herein, showed fully or even the very strong GBSSI activity that weakens, wherein the GBSSI inverted defined gene comprises its 5 ' extra upstream of distinguishing and oppositely copies.Have only 14% usefulness not have the similar construct plant transformed of inverted repeats to have the GBSSI activity that weakens.

Although the mechanism of work of the present invention is understood as yet fully, we believe that the existence of inverted repeats can produce double-stranded RNA, its derive from by the dna sequence dna of reticent dna homolog.Conversely, it is considered to have caused the degraded by the endogenous ssRNA that will be gone out by the genetic transcription of silence.Based on other staff's research ((1998) Plant J 15:737-746 Stam such as Hamilton AJ, M (1997) Annals of Botany 79:3-12), we guess that the inhibition of GBSSI genetic expression is mainly after transcribing.Perhaps, Mette MF etc. ((2000) EMBO J 19:5194-5201) has described the T-DNA that contains the promotor that driving will be transcribed by the inverted repeats of the promotor of the gene of silence (part) (2000 EMBOJ 19:5194-5201 such as Mette) and has caused post-transcriptional silencing and promoter methylation.

The mistake expression level of target gene can be by many factor affecting in the plant.The selection of the transcripting promoter that factor is to use.The compatible promotor of a lot of plants is arranged, comprise tissue-specific and inducible promoter.The composition promotor that speaks well for comprises the arranged in series of CaMV 35S promoter and this promotor, and No.0342926 mentions as european patent application.The other factors that can handle expression level is to transcribe modifying factor, for example intron, polyadenylic acidization and Transcription Termination site.In translation skill, the factor that can consider is the coding proneness of ribosome bind site and gene.The alfalfa mosaic virus that is positioned between promotor and target gene can be improved translation efficiency by the untranslated leader deutero-translational enhancer sequence by the mRNA of coat protein gene of coat protein gene, mentions as US 6037527.Construct of the present invention also can comprise the proteic sequence of one or more coded signals, comprise before-, former-or preceding former-sequence.These are usually located at sequence the place ahead, so target gene and signal protein are expressed as syzygy.Signal sequence can guarantee that any sophisticated syzygy forms fusion product that required posttranslational modification and/or guidance that can be special express and arrives part or organoid in plant or the vegetable cell.

And, observed identical construct expression level difference in plant of inserting sites different in the genome.Believe this result to small part be since gene in chromosomal position, that is, single population has different expression levels.This phenomenon is called " position effect (position effect) " and changes, and is same expression of gene diversity in transformant independently.Use the dna sequence dna of the natural generation of so-called nuclear matrix binding sequence or supporting structure (scaffold) binding sequence to address this problem, in US patent 5773689 and WO 94/24293, propose.WO 0032800 points out that the nuclear matrix binding sequence can improve expression in the floristics of representing monocotyledons and dicotyledons.

In preferred embodiment, the controlling gene expression promoter is strong with tissue-specific.The example of the promotor that known tissue is special comprises the I type potato tuber storage protein promotor (Bevan etc. that are oriented to stem tuber (tuber-directed), (1986) Nucleic Acids Res 14:4625-38), promotor (the 1991 PlantMolec Biol 17 such as Visser of potato tuber GBSSI gene-correlation connection, 691-699), β soybean companion glb promoter of soybean (is also referred to as the proteic promotor of 7S, its driving is oriented to transcribe (Bray 1987 Planta172:364-370) of seed (seed-directed)) and the promotor that is oriented to seed (Pedersen etc., 1982 Cell 29:1015-26) of the zein gene of corn embryosperm.

Perhaps, target gene places transcribing under the control of CaMV 35S promoter.In this makes up, place the tumor-necrosis factor glycoproteins arranged in series of direct motion to improve transcriptional activity by the dna fragmentation that will represent CaMV 35S promoter part.

Select single copy and the transformant that does not contain carrier DNA

After the conversion, T-DNA exists with list or multiple copied in the host plant gene group.And the DNA outside the edge also is incorporated in the genome of host plant sometimes.The situation of this report is, (The Plant J11 is like this in 945-957) for Kononov, ME etc. at the transgenic plant (Martineau, The Plant Cell 6 such as B, 1032-1033,1994) of 20-30% and 75% tobacco transformant.Handle registration authorities that transgenic plant and/or genetically modified food require and think and have selective marker that the transgenic plant of carrier DNA and T-DNA multiple copied/insertion should avoid as far as possible.In the present embodiment, there is not the transformant of skeleton carrier DNA to select by PCR or Southern trace.Perhaps suppress the transfer of carrier DNA to plant by outside the T-border, inserting an encoding sequence, this encoding sequence coding is to plant virose (many) peptide, so there is a negative selection (mentioning as WO 99/01563) to the plant that contains excessive carrier DNA.

Embodiment 1

The optimization that effectively suppresses the gene construct of GBSSI expression

Gene construct

Antisense GBSSI construct pKGBA50 (Fig. 1) describes in (1995) Mol GenGenet 246:745-755 such as Kuipers.Except selectable marker gene NPTII, it is included in the GBSSI cDNA of 2.4Kb of the antisense orientation of GBSSI promotor back.Two GBSSI cDNA fragments, the 5 ' part of 1.1Kb and the 3 ' part of 1.3Kb are cloned into plasmid pWx1.1 and pWx1.3 (1991 Mol Gen Genet 225:289-296 such as Visser) respectively as the EcoRI fragment.These EcoRI fragment clonings advance carrier pMTL25 (1988 Gene 68:139-149 such as Chambers), generate plasmid pMTL1.1 and pMTL1.3, see Fig. 2 and 3.Plasmid pMTL1.3 digests with BamHI.The BamHI fragment cloning of ± 1.3Kb advances the pKGBA50 that BamHI has digested.This has produced two new binary vector: pKGBA50-IR1.3 (Fig. 4), and it has a segmental inverted repeats of 1.3Kb, and pKGBA50-DR1.3 (Fig. 5), contains the segmental forward tumor-necrosis factor glycoproteins of 1.3kb.Plasmid pMTL1.1 digests with SalI.The SalI fragment cloning of ± 1.2kb advances the pKGBA50 that SalI has digested.This has produced two extra binary vector: pKGBA50-IR1.1 (Fig. 6), and it contains the segmental inverted repeats of 1.1kb, and pKGBA50-DR1.1 (Fig. 7), and it has the segmental forward tumor-necrosis factor glycoproteins of 1.1kb.

All constructs all transform into E.coli DH5 α (GIBCO BRL).Binary vector pKGBA50-IR1.3, pKGBA50-IR1.3, pKGBA50-IR1.1, pKGBA50-DR1.1 and pKGBA50-IR1.1 transform into edaphic bacillus LBA4404 (pAL4404) (1983 Nature 303:179-180 such as Hoekema) by the triparental mating method, in E.coli HB101, use helper plasmid pRK2013 (Figurski and Helinski, 1979 Proc Natl Acad Sci USA 76:1648-1652).

Transform potato

Potato cultivar " Karnico " (anonymity, 1994 Beschrijvende Rassenlijstvoor Landbouwgewassen CPRO-DLO, Wageningen, Holland 342pp.) has been transformed antisense constructs pKGBA50 (1995 Mol Gen Genet 246:745-755 such as Kuipers).Identical potato cultivar has transformed pKGBA50-IR1.3 (construct A7), pKGBA50-DR1.3 (construct B 1), pKGBA50-IR1.1 (construct C2) and pKGBA50-DR1.1 (construct D4).The plant of growth in vitro, cultivate altogether by edaphic bacillus with internode cutting and to transform, according to Visser RGF (1991) at K Lindser (ED) plant tissue culture handbook, Kluwer Academic Publishers, Dorrecht/Boston/London, the method for describing among the PP.B5:1-9 is carried out.Contain 20g/L sucrose, selecting transformant in the MS20 substratum of 100mg/L kantlex (Murashige and Skoog, 1962 Physiol Plant 15:473-497).

External stem tuber forms

Branch moves on in the jar that contains 50ml solidified MS30 substratum.3-4 is after week, adds the 20ml liquid nutrient medium, its consist of the KI substratum that contains 325g/L sucrose and 1.75g/L CCC (chlorocholine chloride(ccc), or chlorochline chloride) (' knolinducerend ' (=stem tuber is induced) substratum, Duchefa).Jar placed in 18 ℃ the lucifuge room and grow.2-6 is after week, the test tube potato of having grown on most of branches.

Starch dyeing

Cutting test tube potato is also used iodine solution (1.7% (w/v) tincture of iodine and 3.3% (w/v) liquor kalii iodide) dyeing, the existence of amylose starch in the assessment starch granules.The dyeing microscopic examination of starch granules.

DNA analysis

DNA separates by CTAB DNA separation method from external branch, and as Rogers and Bendich (1998) plant molecular biology manual A6 Kluwer Academic Publ, Dordrecht describes among the pp1-10.Use primer NPT3 and NPT4 (sequence number 3 and 4) to carry out PCR, detect the existence of NPTII selectable marker gene.Subsequently, with primer NPT1 and NPT2, trfA1 and trfA2, insB1 and insB2 (sequence number 1,2,5,6,7 and 8; 1998 MolBreeding 4:343-358 such as Wolters) carry out PCR, the existence of skeleton carrier DNA in the research transformant.

The transformant DNA of 4 μ g digests with HindIII, and fragment is separated with gel electrophoresis, and carries out the Southern Blot experiment.The NPTII probe hybridization that the NPT3+NPT4 PCR product of trace and pBI101 (1987 EMBO J 6:3901-3907 such as Jefferson) is formed detects the quantity that T-DNA inserts.

Reticent efficient

The quantity of each construct transformant has therefrom obtained the test tube potato, is shown in table 1.Tincture of iodine coloration result provides in table 1 and Fig. 9.As a reference, also comprised the result who obtains with antisense constructs pKGBA50.The transformant that 14% usefulness antisense constructs pKGBA50 obtains has shown and has suppressed the GBSSI activity fully, is shown by the red starch granules of dying with iodine solution.Compare with antisense constructs, forward repeats construct pKGBA50-DR1.3 (B1) and pKGBA50-DR1.1 (D4) has produced the reticent fully transformant of low percentage (2-6%).On the contrary, oppositely repeating construct pKGBA50-IR1.3 (A7) and pKGBA50-IR1.1 (C2) has caused higher percentile transformant to show suppressing GBSSI fully, compare with antisense constructs: A7 is 20% and is 62% to C2.Therefore, oppositely repeat construct pKGBA50-IR1.1 (C2) on complete reticent GBSSI activity effective 4.5 times than antisense constructs pKGBA50.It is effectively more than construct A7 oppositely to repeat construct C2: most of (62%) C2 transformant has shown strong or reticent completely, and most of A7 transformant (47%) has shown the silence of medium level.This hint or the zone that is present in encoding sequence in the inverted repeats (5 ' or 3 ' district) are important, and perhaps the direction of the inverted repeats relevant with promotor (antisense DNA-intervening sequence-adopted DNA is arranged to adopted DNA-intervening sequence-antisense DNA is arranged) is effective determinative.Similar, forward repeats to have difference between construct B1 and D4; Most of (51%) B1 transformant has shown low-level silence, and most D4 transformant (85%) shows there is not silence at all.Therefore, the 1.1kb zone of GBSSI cDNA is the most effective to silence when reverse repetition direction, and it is the most invalid when the forward repetition direction.This hint is sought the suitableeest sequence and is removed to construct reverse repetition construct to produce maximum reticent effect be very important.

Analysis of molecules

DNA isolation from 20 A7 transformants.The NPTII PCR fragment that all have all shown expection shows that all contain at least one T-DNA and insert.There are 9 to show and have non-T-DNA carrier DNA in 20, by the PCR decision (table 2) of NPTIII and trfA gene.Therefore, only about half of transformant does not contain skeleton carrier DNA.

DNA isolation from 40 C2 transformants that shown strong complete reticent GBSSI.Use the NPTII primer, all have all shown the PCR fragment of expection.With the pcr analysis of skeleton carrier dna primer proof wherein 17 transformants contain non-T-DNA sequence, and 23 be negative (table 3).The DNA of HindIII digestion and the Southern engram analysis of NPTII probe hybridization show has 5 to contain strand T-DNA insertion in 20 transformants.Wherein 4 do not have skeleton carrier DNA.Therefore, this proof may obtain to have the transformant that strand T-DNA inserted and do not have the complete silence of skeleton carrier DNA with oppositely repeating construct pKGBA50-IR1.1.

Embodiment 2

The transformant that separates the GBSSI silence that does not have selectable marker gene

Gene construct

The 1.1kb zone that embodiment 1 shows GBSSI cDNA reverse repetition direction to silence be the most effective and may with this construct (pKGBA50-IR1.1) acquisition have strand T-DNA and do not have skeleton carrier DNA complete silence transformant.

Among the former embodiment, on kantlex, select transformant with the selectable marker gene NPTII that is present on the construct pKGBA50-IR1.1.For separation does not have the transformant of selectable marker gene, binary vector pKGBA50-IR1.1 is with enzyme PmeI and ClaI digestion (see figure 1).PmeI produces flat terminal.The ClaI sticky end is treated to flat terminal with the Klenow polysaccharase, carrier DNA carries out cyclisation with the T4 dna ligase by flat terminal the connection then.This has produced carrier pKGBA50mf-IR1.1 (not having marker gene) (Fig. 8).This construct transforms into E.coli DH5 α (GIBCO BRL).Binary vector pKGBA50mf-IR1.1 transforms into edaphic bacillus LBA4404 (pAL4404) (Hoekema etc. 1983) by triparental mating, in E.coli HB101, use helper plasmid pRK2013 (Figurski and Helinski, 1979) and edaphic bacillus strain Agl-0 (Lazo etc. 1991).Known Agl-0 bacterial strain is bigger than LBA4404 toxicity.

Transform potato and select transformant

The cutting of the internode of the plant of the growth in vitro of potato cultivar Karnico is used for cultivating altogether by edaphic bacillus and transforms, the method for describing according to Visser (1991).Potato cultivar Karnico has transformed the pKGBA50mf-IR1.1 of edaphic bacillus LBA4404 or edaphic bacillus Agl-0, according to identical method.Do not select.After 4 weeks, gather in the crops first branch and gathered in the crops branch 3 months continuously.Each stem explant has been gathered in the crops and has been no more than two regenerates and branch growth in the MS20 substratum.1-2 is after week, collects the leaf of 8 or more independently branches or stem material and is created as storehouse (size in storehouse depends on the frequency of the transformant of expection).The DNA in these storehouses of branch uses the Magnesil genomic dna separating kit available from Promega to be separated in the 96 hole enzyme plates.Use primer BINMCS the DNA that separates from the regenerate storehouse to be carried out pcr analysis, detect the existence of transformant with GBSS-0 (sequence number 9 and 10).The leaf in the positive storehouse of each PCR and/or stem are collected in the 96 hole enzyme plates and use Magnesil genomic dna separating kit isolation of genomic DNA available from Promega.With GBSS-0 (sequence number 9 and 10) DNA that separates from each regenerate is carried out pcr analysis with primer BINMCS, detect the existence of transformant.

External stem tuber forms

To the PCR positive branches, add the 20ml liquid nutrient medium, its consist of the KI substratum that contains 325g/L sucrose and 1.75g/L CCC (chlorocholine chloride(ccc), or chlorochline chloride) (knolinducerend (=stem tuber is induced) substratum, Duchefa).Place 18 ℃ lucifuge room to grow in jar.After 2 to 6 weeks, formed the test tube potato on most of branch.

Starch dyeing

Cutting test tube potato and with the existence of amylose starch in the iodine solution dyeing assessment starch granules.The dyeing microscopic examination of starch granules.

DNA analysis

With the DNA of the outer branch of CTAB DNA separation method chorista, such as Rogers and Bendich (1998) description.With primer NPT1 and NPT2, trfA1 and trfA2, insB1 and insB2 (sequence number 1,2,5,6,7 and 8; 1998 Mol Breeding 4:343-358 such as Wolters) carry out PCR, the existence of skeleton carrier DNA in the research transformant.The DNA of 4 μ g transformants digests with HindIII, and the gel electrophoresis isolated fragment also carries out the Southern Blot experiment.Trace and NOS stop probe hybridization, detect the quantity that T-DNA inserts.

Produce adventitious shoot (adventitious shoot)

According to (1998) such as Hovenkamp-Hermelink, can produce does not have marker gene, does not have amylose starch, does not have carrier DNA and does not have genotypic adventitious shoot.The leaf of the branch of each genotypic 10 to 20 sterile culture is at the 147mg/lCaCl that has added 10mg/l BAP and 10mg/l NAA ₂2H ₂O and 80mg/l NH ₄NO ₃Solution in floating.After the floating evening (16 hours), leaf is cut to the wide explant of 3-4mm, and 100 explants of each genotype also place the callosity that contains 40g/l N.F,USP MANNITOL, 10g/l sucrose, 2.25mg/l BAP, 0.0175mg/l IAA and 8g/1 agar to induce the MS substratum.After 6 days, explant is transferred to and has been added 15g/l sucrose, 2.25mg/lBAP, 5mg/l GA ₃With regenerate in the MS substratum of 8g/l agar.Per 3 weeks of explant are transferred in the fresh regeneration culture medium.Collect adventitious shoot (each explant 2-4), each genotype is collected 100 branches at least and is grown in the container of the MS substratum that contains 20g/l sucrose.When branch had formed more than 4 joints, they were used for the starch screening that aforesaid external stem tuber formed and do not have amylose starch.

The efficient that does not have mark to transform

At 5 independently in the experiment, approximately the pKGBA50mf-IR1.1 of 8000 stem explants of potato cultivar Karnico and edaphic bacillus LBA4404 or AGL-0 is hatched.The inoculation back is after 1 to 4 months, and each explant is collected 1 or 2 regenerated branch and grown on Murashige and Skoog substratum.After 1 to 2 week, collect at least 8 independently the branch leaf or stem material and build the storehouse.The DNA in these storehouses of branch is separated in the 96 hole enzyme plates.With polymerase chain reaction (PCR) is the method on basis, and we have detected the existence of the transformant in the branch storehouse.In the positive storehouse of PCR, analyze from the DNA of each aftergrowth separating, to detect existing of transformant.After LBA4404 transformed, the branch that is less than 0.2% collection was the PCR male, and this percentage that AGL-0 transforms is 4.6% (table 4).5 independently the frequency of the positive transformant of PCR of AGL-0 transformation experiment is 1.3 and 5.6%, and this changes between 0 to 0.8% to LBA4404.

Show for determining the required frequency that does not have the PCR positive branches of amylose starch phenotype, branch growth in vitro under the high-sucrose condition to induce stem tuber.The test tube potato of having grown is with the existence by the microscopic evaluation amylose starch of tincture of iodine dyeing and starch granules.In 220 PCR male transformants being analyzed, about 45% shows and to have suppressed the GBSSI activity fully, is shown by the starch granules of redness.This per-cent is not still than having the low of amylose starch with NptII as 60% the plant that the pKGBA50mf-IR1.1 of selective marker obtains, and accidental we of this hint may select false positive with the PCR detection system.

Yet the result has effectively shown without marker gene and selects the feasibility of transformant and efficient that acquisition does not have the mark transformant to improve by the gene silencing construct that uses toxicity soil bacterial strain and optimized.

Select singly to copy and do not have the transformant of carrier DNA

Because all arguements in view of DNA integrates except interested transformant gene, should also separate the transformant that does not have carrier DNA and the T-DNA that insert more.The genome that DNA is integrated into the host plant outside the border it is reported and takes place in 20 to 75% plant transformed.By having used at the pcr analysis of the primer of 5 open reading frames of pBIN19 carrier not having mark, not having the existence of skeleton carrier DNA in the Transformation of potato body of amylose starch of selecting.39 is negative to 5 dna fragmentations in the transformant that does not have amylose starch of 99 detections.These 39 transformants that do not have carrier DNA are further by analyzing (Figure 10) with the NOS terminator as the Southern blot hybridization of probe.These are analyzed and show that (30) transformant contains 3 or the still less T-DNA insertion of copy number mostly.Yet 14 contain strand T-DNA insertion in 39 transformants of analysis.

Therefore, this has proved the feasibility that does not have the transformant of skeleton carrier DNA without the selectable marker gene acquisition, and this transformant only contains the T-DNA of an insertion, and wherein native gene is by reticent fully.

Mosaic

For studying transformant that PCR whether selects is mosaic from a plurality of cell regenerationes, and we use the padding of the synthetic iodine solution that suppresses all the time of amylose starch with the stem tuber of cutting.Each independently in the stem tuber dyeing course of transformant, does not observe fan-shaped dyeing.Institute's tuberosity or complete red or full indigo plant.To 60 transformants independently, the dyeing that each transformant surpasses 20 stem tubers shows to have only a cording that red and blue segmental cell are arranged with the tincture of iodine.These results show chimeric the generation, yet frequency is very low.And then, use the adventitious shoot method that 28 are not independently had mark, do not have amylose starch and do not have the transformant of carrier DNA to carry out chimeric screening.Each genotype has produced that the adventitious shoot that surpasses 100 and each are used for all that external stem tuber forms and screening does not have the phenotype of amylose starch.Screened above behind 3000 branches, we do not find an adventitious shoot that has formed the test tube potato that contains amylose starch.This method for transformation that does not have mark that proves us or with very low ratio does not produce chimeric plant.

Embodiment 3

Method for transformation is to obtaining not have the influence of mark transformant efficient

For determining method for transformation, two method for transformation have been tested to obtaining not have the influence of mark transformant efficient.

Potato cultivar Karnico has transformed the explant that has the Agrobacterium tumefaciens AGL0 that contains plant conversion carrier pKGBA50mf-IR1.1 by cultivating altogether.Method for transformation 1.1 (Visser, 1991)

Potato cultivar Karnicode branch point is cultivated under pH5.6 in the substratum that is containing the main and less important salinity of MS, 3% sucrose, 0.8% agar.Culture is at 21 ℃, and 16 hour photoperiod be 4 weeks of growth down.The internode of stem is cut into 2 to 5mm long and before cultivating altogether, on the two layers of filter paper that is placed on the solid R3B substratum, placed 1 day.The R3B substratum that uses contains the salinity and the VITAMIN of MS (4.71g/l) substratum, and has added 3% sucrose, 2mg/l NAA, 1mg/lBAP and 0.8% agar, pH5.8.The pH that filter paper layer is coated with 2ml is 6.5 MS (4.71g/l), 2.0g/l caseic hydrolysate, 3% sucrose, 1mg/l2, the PACM liquid nutrient medium that 4D and 0.5mg/L phytokinin are formed.Cultivated altogether 5 to 10 minutes with the edaphic bacillus of cultivating two days, then the explant trace to the filter paper to remove unnecessary bacterium.After finishing trace, explant shifts back same culture dish and at 21 ℃, hatched two days under 16 hour photoperiod.

Two days later, explant is transferred in the Zcvh substratum of 4.71g/l MS, 2.0% sucrose, 0.8% agar, 200mg/l cefotaxime sodium, 200mg/l vancomycin and 1mg/l corn glycosides composition.Per two weeks of culture are transferred to fresh Zcvh substratum.4 all backs are collected first and are transferred in the substratum that contains the main and less important salinity of MS, 3% sucrose, 0.8% agar, pH5.6.Each explant is collected to be no more than 2 branches and to collect and is continued about 3 months.Method for transformation 2 (Plant Molec Biol 17:89-100 such as Edwards, 1991)

The branch point of potato cultivar Karnico is cultivated in the substratum that contains the main and less important salinity of MS, 3% sucrose, 0.8% agar, pH5.6.Culture is at 21 ℃, and 16 hour photoperiod be 4 weeks of growth down.The internode of stem is cut into 2 to 5mm long and before cultivating altogether, on the two layers of filter paper that is placed on the solid R3B substratum, placed 1 day.The R3B substratum that uses contains the salinity and the VITAMIN of MS (4.71g/l) substratum, and has added 3% sucrose, 2mg/l NAA, 1mg/lBAP and 0.8% agar, pH5.8.The pH that filter paper layer is coated with 2ml is 5.8 MS (4.71g/l), 2.0g/l caseic hydrolysate, 3% sucrose, 1mg/l 2, the PACM liquid nutrient medium that 4D and 0.5mg/L phytokinin are formed.Cultivated altogether 30 minutes with the edaphic bacillus of cultivating two days, then the explant trace to the filter paper to remove unnecessary bacterium.After finishing trace, explant is transferred to 4.71g/l MS, 2.0% sucrose, 0.8% agar, 2mg/l 2, and 4D and 0.5mg/l corn glycosides are formed, and in the solid PCM substratum of pH5.8 and under 21 ℃, hatch in the dark two days.Two days later, explant is transferred in the PCM substratum that contains the 200mg/l cefotaxime sodium, at 21 ℃, hatches 4 days under 16 hour photoperiod.

After such 4 days, explant is transferred to 4.71g/l MS, 2.0% sucrose, 0.8% agar, 2mg/lGA ₃, in the solid PSM substratum formed of 1.0mg/l corn glycosides, pH5.8 and 200mg/l cefotaxime sodium.Per three weeks are transferred to culture in the fresh PSM substratum that contains the 200mg/l cefotaxime sodium.All around, collect first and be transferred in the substratum that contains the main and less important salinity of MS, 3% sucrose, 0.8% agar, pH5.6.Each explant is collected to be no more than two branches and to collect and is continued about 3 months.

After 1 to 2 week, collect the leaf or the stem material of 8 or more independently branch and build storehouse (the storehouse size depends on the frequency of expection transformant).Use Magnesil genomic dna separating kit to be separated in the 96 hole enzyme plates DNA in these storehouses of branch available from Promega.With primer BINMCS and GBSS-0 (sequence number 9 and 10) DNA that separates from the storehouse of regenerate is carried out pcr analysis, to detect existing of transformant.Each the leaf in the positive storehouse of PCR and/or stem material are collected in the 96 hole enzyme plates and use Magnesil genomic dna separating kit isolation of genomic DNA available from Promega.With GBSS-0 (sequence number 9 and 10) DNA that separates from each regenerate is carried out pcr analysis with primer BINMCS, to detect existing of transformant.

The result

Use the 1000 stem explants of method for transformation 1 and 2 couples of potato cultivar Karnico to carry out two independently conversions.The frequency of therefrom collecting the explant of branch is comparable between two method for transformation.Method 1 has produced 74% to be had the explant of branch and has 67% explant to form one or more with method 2.The frequency of the PCR positive branches that obtains is very different in two kinds of methods.With method 20.6% to 1.0% collect the branch is the PCR male, and with method 1 this per-cent between 5.5% to 6.0%.

Therefore, use optimized method for transformation can produce the PCR positive branches of 5 to 10 multiple amounts and the efficient that raising does not have the conversion of mark.

Embodiment 4

There is not mark ground to select the strain of high-lysine Transformation of potato

Gene construct

Feed back insensitive potato DHPS and generate for halfcystine, mention as EP 99204520 by the 134 amino acids residues (l-asparagine) that change evolution conservative.Structure contains the mosaic gene of mutant DHPS gene, at first with DHPS cDNA from pTriplex carrier (pAAP42) be cloned in pCR-Script SK (+) (pAAP55) again from this carrier with its as the XbaI-EcoRI fragment cloning the pBluescript SK carrier that XbaI-EcoR has digested (pAAP57).5 ' end, the DHPS cDNA of sudden change have been fused to the HindIII-SaII fragment of the GBSSI promoter fragment of 800bp length.The downstream of sudden change DHPS sequence, the termination signal of the rouge alkali synthetase gene of Agrobacterium tumefaciens is inserted (Greve, H.D. etc. (1983) J.Mol.Appl.Genet.1:499-511) as the SstI-EcoRI fragment.Whole mosaic genes is cloned the into HindIII-EcoRI site of pBINPLUS, pBINPLUS is digested by ClaI earlier and RcaI partly digests and be connected to remove NPTII selective marker (Van Engelen, F.A. etc. (1995) TransgenicResearch 4:288-290) again.This has produced carrier pAAP105mf (Figure 10).All constructs all transform into E.coli DH5 α (GIBCO BRL).

Transform potato plant

Binary vector pAAP 105mf is used for carrying out freezing thawing and transforms Agrobacterium tumefaciens strain AGL0 (Hofgen, R and Willmitzer, L (1988) Nucl.Acids Res.16:9877).The AGL0 that has transformed is used to inoculate potato (solanum tuberosum, Karnico kind) stem explant subsequently.Do not select.All around, collect first and collection continue about three months.Each stem explant is collected and is no more than two regenerates and branch growth in the MS20 substratum.After 1 to 2 week, collect the leaf or the stem material of 8 or more independently branch and build storehouse (the storehouse size depends on the frequency of expection transformant).DNA in these storehouses of branch uses the Magnesil genomic dna separating kit available from Promega to be separated in the 96 hole enzyme plates.With GBSS-0 (sequence number 9 and 10) DNA that separates from the regenerate storehouse is carried out pcr analysis with primer BINMCS, to detect existing of transformant.Each, the leaf in the positive storehouse of PCR and/or the stem material is collected in the 96 hole enzyme plates and genomic dna uses the Magnesil genomic dna separating kit available from Promega to separate.With GBSS-0 (sequence number 9 and 10) DNA that separates from each regenerate is carried out pcr analysis with primer BINMCS, to detect existing of transformant.

External formation stem tuber

To the PCR positive branches, add the 20ml liquid nutrient medium, it consists of the KI substratum.Jar is placed in the growth room of the dark under the room temperature.After 2 to 4 weeks, most of branch has been grown the test tube potato.

The analysis of free aminoacid content in the transgenic plant

The homogenate tissue (0.5-1.0 gram) in the 50mM of the 2ml that contains 1mM dithiothreitol dithio Pi damping fluid (pH7.0) with mortar and mallet.Add nor-leucine as internal reference.Total free aminoacids 5ml water: chloroform: carbinol mixture (3: 5: 12, volume ratio) extraction carrying out partial purification.Collect water, organic phase is extracting twice again.After being concentrated into 3ml with lyophilization, 20 microlitre samples use cationic exchange coloum to analyze by HPLC, with the three bronze medal derivatization methods of indenes behind the amino acid whose post in 570nm and 440nm detection (BIOCHROM 20, Amersham Pharmaciabiotech).Figure 11 has shown that Methionin does not have to account in the stem tuber of the potato plant that transforms and 17 potato plants that contain sudden change DHPS gene construct pAAP105mf the per-cent of total amino acid 12 contrasts.Lysine level 2-2.5% from contrast wild-type potato is increased to maximum 30 per-cents in transforming stem tuber, has caused Methionin to become the indispensable amino acid of " great majority " amino acid rather than " low-level ".

Embodiment 5

There is not the unmarked selection of the high-lysine Transformation of potato strain of amylose starch

Gene construct

Synthesized the connexon that contains at the compatible overhang (overhang) of 5 ' the end HindIII-overhang compatible with 3 ' end EcoRI-

(5 '-AGCTGCATATGAAGCTTTCTAGATCTGAATTCCATATGT-3 ' and 3 '-CGTATACTTCGAAAGATCTAGACTTAAGGTATACATTAA-5 ').This connexon is connected on the pUC28 plasmid of HindIII/EcoRI digestion (1993 Gene130:151-152 such as Benes), has produced plasmid pAAP171.Construct pAAP105, the binary vector that contains sudden change DHPS gene digests with HindIII and EcoRI.The fragment cloning that contains the 2.2kb of GBSSI promotor-DHPS gene-NOS terminator advances among the pAAP171 of HindIII/EcoRI digestion.This has produced construct pAAP172 (seeing Figure 12).

Construct pAAP172 digests with NdeI and ScaI (the ScaI site is present in the pUC28 skeleton).The DNA that has digested uses ethanol sedimentation again with phenol/chloroform extraction.Binary vector pKGBA50mf-IR1.1 digests with VspI, uses alkaline phosphatase treatment again, phenol/chloroform extraction, ethanol sedimentation.The NdeI fragment of the 2.2kb of PAAP172 connects the into pKGBA50mf-IR1.1 carrier (Figure 13) of VspI digestion, two different constructs: pDHPS-IR1.1 (series connection) have been produced, wherein two GBSSI promotors are pointed to same direction (Figure 14), and pDHPS-IR1.1 (oppositely), wherein two GBSSI promotor directed in opposite directions (Figure 15).These constructs transform into Agrobacterium tumefaciens Agl-0 from E.coli DH5 α.

Transform the selection of potato and transformant

The internode cut substrate of the growth in vitro plant of potato cultivar Kamico transforms by cultivating altogether with Agrobacterium tumefaciens, according to the method for Visser (1991) description.

Potato cultivar Kamico has transformed pDHPS-IR1.1 (series connection) and pDHPS-IR1.1 (oppositely) among the Agrobacterium tumefaciens Agl-0 with identical method.Do not select.All around, collect first branch and continue about three months of collection.Each stem explant has been collected and has been no more than two regenerates and branch growth in the MS20 substratum.After 1 to 2 week, collect 8 or more independently the branch leaf or stem material and build the storehouse (the storehouse size depend on the expection transformant frequency).DNA in these storehouses of principle uses the Magnesil genomic dna separating kit available from Promega to be separated in the 96 hole enzyme plates.With GBSS-0 (sequence number 9 and 10) DNA that separates from the regenerate storehouse is carried out pcr analysis with primer BINMCS, to detect existing of transformant.Collect each the leaf in the positive storehouse of PCR and/or the stem material in 96 hole enzyme plates and use Magnesil genomic dna separating kit isolation of genomic DNA available from Promega.With GBSS-0 (sequence number 9 and 10) DNA that separates from each regenerate is carried out pcr analysis with primer BINMCS, to detect existing of transformant.

External formation stem tuber

To the PCR positive branches, add the 20ml liquid nutrient medium, it consists of the Kl substratum.Jar places the growth room of the dark under 18 ℃.After 2 to 4 weeks, most of branch has been grown the test tube potato.

Starch dyeing

Cutting test tube potato and with iodine solution dyeing existing with amylose starch in the assessment starch granules.The dyeing microscopic examination of starch granules.

The analysis of free aminoacid content in the transgenic plant

The homogenate tissue (0.5-1.0 gram) in containing the 2ml 50mM Pi damping fluid (pH7.0) of 1mM dithiothreitol dithio with mortar and mallet.Add nor-leucine as internal reference.Total free aminoacids 5ml water: chloroform: carbinol mixture (3: 5: 12) extraction carrying out partial purification.Collect water, remaining extracting twice again.After being concentrated into 3ml with lyophilization, 20 microlitre samples use cationic exchange coloum to analyze by HPLC, with the three bronze medal derivatization methods of indenes behind the amino acid whose post in 570nm and 440nm detection (BIOCHROM 20, Amersham Pharmacia biotech).

Embodiment 6

There is not the unmarked selection of the cassava transformant of amylose starch

The step of converting of having described the Agrobacterium tumefaciens mediation with the lower section is to produce the cassava plant of genetic modification, and its starch mainly consists of amylopectin, and does not have selectable marker gene such as nptII, pat, basta, htp or other is auxiliary.

Gene construct

Design PCR primer (sequence number 12-20) is with amplification cassava GBSSI cDNA sequence part: two nested forwards and a reverse primer are used for following three zones: 5 ' partly (1-800 base), middle portion (800-1500 base) and 3 ' partly (1200-2000 base).Forward primer contains the EcoRI restriction site, and reverse primer contains the XbaI restriction site.The PCR product of each of three parts of GBSSI cDNA all digests with EcoRI and is connected.Gained connects the part inverted repeats that product contains cassava GBSSI cDNA, and the sequence between two nested forward primers is as transcribed spacer.This sequence is advanced carrier pMTL24 (Chambers etc. 1988) with XbaI digestion and clone.Inverted repeats is with BamHI digestion and remove from this carrier, connects the NOS terminator (see figure 8) of the binary vector pKGBA50mf-IR1.1 that potato GBSSI promotor and BamHI digested subsequently.Like this, the cassava GBSSI that has obtained not have three marker gene oppositely repeats construct.These transform into Agrobacterium tumefaciens strains A gl-0.

The vegetable material and the culture medium for tissue culture that use

The cultivation of TMS60444 and Adira4 plant be every month with the cut substrate subclone of a node to replenished Murashige and Skoog (1962, Physiol.Plant.15:473-497) in the substratum of salt and VITAMIN and 40g/l sucrose (MS4).Fragile embryo's generation callus (friableembryogenic callus (FEC)) is following generation:

-separation meristem or prematurity leaf from the donor plant

-go up cultivation meristem/prematurity leaf at the MS40 that has replenished 6mg/l NAA and 6mg/l picloram (Picloram)

-separate complete embryo take place tissue and replenished Gresshoff and Doy (1974, Planta107:161-170) salt and VITAMIN, the substratum (GD6) of 60g/l sucrose and 10mg/l picloram

The middle cultivation

-separate FEC (fritter accumulative, spherical unit), in the GD6 substratum, cultivate.FEC per 3 weeks in the GD6 substratum carry out subculture and keep.Schenk and Hildebrandt (1972 have been replenished by transferase 10 .5gFEC to containing 50ml, Canadian Journal of Botany 50:199-204) salt and VITAMIN, the 200ml flask of the liquid nutrient medium (SH6) of 60g/l sucrose and 10mg/l picloram produces liquid culture.Substratum changes weekly twice and the inclusion in 2 each flask of all backs is assigned in 5 new flasks.Flask is gone up at shaking table (LAB-line InstrumentsInc.Model 3519) and is cultivated with 120rpm.

Infect FEC with edaphic bacillus

The FEC sample of the TMS604444 of 100mg is as parent material.FEC is dispersed in the substratum that has replenished Gresshoff and Doy salt and VITAMIN, 60g/l sucrose, 10g/l agar and 10mg/l picloram, and adds several and contain the edaphic bacillus strains A gl-0 that GBSSI oppositely repeats construct.Two days later, wash twice of FEC to remove unnecessary edaphic bacillus with sterile distilled water.After inferior, FEC transfers in the liquid nutrient medium (SH6) that contains Schenk and Hildebrandt salt and VITAMIN, 60g/l sucrose, 10g/l agar and 10mg/l picloram, and it has replenished microbiotic such as cefotaxime sodium to kill edaphic bacillus.One week back renewal substratum, careful being dispersed in of two week back FEC replenished in the GD6 substratum of cefotaxime sodium.After two weeks, 10 50mg FEC that infect sample use PCR to estimate genetically modified whole and degree as the selective system on basis.Collect small portion FEC material and use DNA available from the Magnesil genomic dna separating kit isolated cell of Promega.With primer BINMCS and GBSS-0 (sequence number 9 and 10) the DNA isolate is carried out pcr analysis, to detect existing of transformant.There are 2 to be GBSS-0/BINMCS primer male in 10 samples.These sample cultivation are in the maturation medium of having replenished cefotaxime sodium.Maturation medium consists of Murashige salt and VITAMIN, 40g/l sucrose, 10g/l agar (MS4) and 1mg/l picloram.Tissue was cultivated four months altogether every being transferred in two weeks in the fresh maturation medium.Therebetween, the somatic embryo that separates the torpedo setting that comes from the FEC growth is also cultivated the MS4 substratum that has replenished 0.1mg/l BAP and cefotaxime sodium to allow it develop into sophisticated somatic embryo.Sophisticated somatic embryo is at first cultivated a week in the liquid MS4 that has replenished 1.0mg/l BAP and cefotaxime sodium, is transferred to then in the solid MS4 substratum that has replenished 1.0mg/l BAP.The branch of somatic embryo development is taken root in the MS4 that has replenished cefotaxime sodium.All plants that take root are used for the selective system of PCR for the basis.Collect the leaf material of 8 branches and build the storehouse.DNA in these storehouses of branch uses the Magnesil genomic dna separating kit available from Promega to be separated in the 96 hole enzyme plates.With GBSS-0 (sequence number 9 and 10) DNA that separates from the regenerate storehouse is carried out pcr analysis with primer BINMCS, to detect existing of transformant.Collect each the leaf in the positive storehouse of PCR and/or the stem material in 96 hole enzyme plates and use Magnesil genomic dna separating kit isolation of genomic DNA available from Promega.With GBSS-0 (sequence number 9 and 10) DNA that separates from each regenerate is carried out pcr analysis with primer BINMCS, to detect existing of transformant.Altogether, system has obtained 1264 and 984 plant from two kinds, and it is PCR male that 5 and 38 plant are wherein arranged respectively.The PCR positive plant is grown in the canister in greenhouse.After three months from stem tuber separating starch and analyze the content of amylose starch.In 27 plant, there is not amylose starch substantially.

Table 1. is by the reticent level of the transformant of different constructs acquisitions

The reticent level of GBSSI

The quantity of construct transformant

Fully/weak nothings such as persistent erection

C2????????????133??????????83(62％)?????6(5％)?????2(2％)??????42(31％)

A7????????????118??????????24(20％)?????55(47％)???10(8％)?????29(25％)

D4????????????66???????????4(6％)???????2(3％)?????4(6％)??????56(85％)

B1????????????77???????????2(2％)???????7(9％)?????39(51％)????29(38％)

Antisense 144 20 (14%) 20 (14%) 22 (15%) 82 (57%)

The analysis of molecules of each A7 transformant of table 2.

The PCR fragment

The reticent level of transformant

NPTII????NPTIII????????trfA

A little less than the A7-1+--

A7-3 do not have+--

A little less than the A7-7+--

A7-11 do not have+--

A7-14 is strong+++

A7-22 is strong+++

A7-26 is strong+++

A7-32 is strong+--

A little less than the A7-41+--

A7-42 is strong+++

A7-43 is strong+++

A7-44 is strong+--

A little less than the A7-48+--

A7-53 is strong+++

A7-62 is medium+++

A7-74 is strong+--

A little less than the A7-76+--

A7-84 is strong++-

A7-95 fully++-

A little less than the A7-98+--

The analysis of molecules of each C2 transformant of table 3.

The PCR fragment

T-DNA

The reticent level of transformant

NPTII NPTIII trfA InsB band quantity

C2-1 is strong++ ND ND 4

C2-5 is medium++--ND

A little less than the C2-6+---ND

C2-7 fully++ ND ND＞5

C2-8 fully++ ND ND＞5

C2-10 is strong++ ND ND 3

C2-11 fully+++ND＞5

C2-14 fully+--ND 3

C2-15 fully+--ND 4

C2-16 is medium+--ND 2

C2-17 fully+? + ND 3

C2-20 fully+--ND 1

C2-25 fully++ ND ND 2

C2-29 fully+--ND 1

C2-30 fully++ ND ND＞4

C2-33 is strong+---ND

C2-38 is strong+--ND 1

C2-41 is strong+---ND

C2-43 is strong+++-ND

C2-47 fully+-+-ND

C2-65 fully++ ND ND＞4

C2-66 fully+--ND 2

C2-70 is strong+---ND

C2-71 fully+--ND 2

C2-75 is strong+---ND

C2-80 is strong+---ND

C2-83 is strong++ ND ND 1

C2-85 is strong++++ ND

C2-86 is medium+---ND

C2-95 is strong+---ND

C2-112 is strong+++-ND

C2-114 fully+--ND 1

C2-116 is strong++ ND ND 4

C2-151 is strong+---ND

C2-152 is strong+---ND

C2-156 fully+---ND

C2-167 is strong++-+ND

C2-168 is strong+---ND

C2-170 is strong+---ND

C2-180 is strong+---ND

The ND=undetermined

Table 4. edaphic bacillus bacterial strain is to the influence of Transformation of potato efficient

	Pcr analysis	Phenotype analytical
				The PCR positive branches a	No amylose starch transformant b
??Exp.1，LBA4404 ??AGL-0	????0/1112(0.0) ????50/888(5.6)	??0/0(0.0) ??17/40(42.5)
			??Exp.2，LBA4404 ??AGL-0	????3/632(0.5) ????10/420(2.4)	??1/3(33.3) ??8/10(80.0)
??Exp.3，LBA4404 ??AGL-0	????0/440(0.0) ????31/651(4.8)	??0/0(0.0) ??10/31(32.3)
			??Exp.4，LBA4404 ??AGL-0	????2/240(0.8) ????9/688(1.3)	??2/2(100) ??2/9(22.2)
??Exp.4，AGL-0	????128/2370(5.4)	??60/125(48.0)
			The total quantity of LBA4404 regeneration branch	????5/2424(0.2)	??3/5(60.0)
The total quantity of AGL-0 regeneration branch	????228/5017(4.6)	??97/215(45.1)

A is by the quantity (%) of the PCR positive branches that obtains of sum of the branch of test

The quantity (%) of the no amylose starch transformant that b is obtained by the sum of transformant of test

Primer sequence

Sequence number 1 (NPT1):

5’-TCC?ACC?TTA?TCG?GCA?ATG?AA-3′

Sequence number 2 (NPT2):

5’-CGG?CAG?TGA?GAG?CAG?AGA?TA-3′

Sequence number 3 (NPT3):

5’-TCG?GCT?ATG?ACT?GGG?CAC?AAC?AGA-3′

Sequence number 4 (NPT4):

5’-AAG?AAG?GCG?ATA?GAA?GGC?GAT?GCG-3′

Sequence number 5 (trfA1):

5’-TTC?TCC?TCG?TGC?TCG?TAA?AC-3′

Sequence number 6 (trfA2):

5’-GGT?CGC?TGG?TAT?TCG?TGC-3′

Sequence number 7 (insB1):

5’-GCG?CTA?TCT?CTG?CTC?TCA?CT-3′

Sequence number 8 (insB2):

5’-AAC?GGC?CTC?ACC?CCA?AAA?A-3′

Sequence number 9 (BINMCS):

5’-GCA?CCC?CAG?GCT?TTA?CAC?TT-3’

Sequence number 10 (GBSS-0):

5’-TAC?CGC?TAC?CAC?TTG?ACA?TTC-3’

Sequence number 11:

5’AGCT???GCATATGAAGCTTTCTAGATCTGAATTCCATATGT?3’

3’??CGTATACTTCGAAAGATCTAGACTTAAGGTATACATTAA??5’

Sequence number 12:

Primer?5CASGBF1：5’-TAG?AAT?TCA?CCA?GCG?GAA?CCT?ATT?TT-3’

Sequence number 13:

Primer?5CASGBF2：5’-ATG?AAT?TCG?GAC?CCA?AAC?TAT?CAC?TC-3’

Sequence number 14:

Primer?5CASGBR1：5’-TGT?CTA?GAA?TGG?AAG?CAG?AGC?AGT?GT-3’

Sequence number 15:

Primer?MCASGBF1：5’-CAG?AAT?TCA?AGC?CAT?TTA?CCA?ACC?TA-3’

Sequence number 16:

Primer?MCASGBF2：5’-ATG?AAT?TCT?ATG?AGA?AGC?CCG?TGA?AG-3’

Sequence number 17:

Primer?MCASGBR1：5’-CAT?CTA?GAG?AAC?CAG?CAT?AAA?GTC?AG-3’

Sequence number 18:

Primer?3CASGBF1：5’-TAG?AAT?TCG?GCT?TCA?TTG?GTA?GAT?TA-3’

Sequence number 19:

Primer?3CASGBF2：5’-TAG?AAT?TCA?TTG?AGC?ATC?TGG?AGG?TT-3’

Sequence number 20:

Primer?3CASGBR1：5’-TAT?CTA?GAC?CAT?TCA?CCC?TTC?ACA?AA-3’

Claims (30)

1. recombinant nucleic acid that comprises the T-DNA construct, it allows described construct to shift to enter in the vegetable cell genome, and described construct has a kind of exogenous nucleic acid that does not contain the nucleic acid of the selective marker of encoding.

2. the nucleic acid of claim 1, wherein said T-DNA construct comprises at least one T-DNA border.

3. claim 1 or 2 nucleic acid, there is T-DNA border tumor-necrosis factor glycoproteins in wherein said exogenous nucleic acid flank.

4. each nucleic acid in the claim 1 to 3, wherein said T-DNA derives from the Ti-plasmids of edaphic bacillus spp.

5. the nucleic acid of claim 4, wherein said edaphic bacillus comprises Agrobacterium tumefaciens.

6. each nucleic acid in the claim 1 to 5, wherein said exogenous nucleic acid allow to regulate and control target gene expression in described genome.

7. the nucleic acid of claim 6, wherein said regulation and control comprise downward modulation.

8. the nucleic acid of claim 7, wherein said exogenous nucleic acid comprises the inverted repeats to the small part polynucleotide region of described target gene.

9. each nucleic acid in the claim 6 to 8, wherein said target gene coding starch small grain constraint starch synthase (GBSSI).

10. each nucleic acid in the claim 1 to 5, wherein said exogenous nucleic acid allows expressing heterologous polypeptide in described vegetable cell.

11. the nucleic acid of claim 10, wherein said heterologous polypeptide comprises a kind of enzyme.

12. the nucleic acid of claim 11, wherein said enzyme comprises dihydro-2, and dipicolimic acid 2 synthase (DHPS) is preferably feedback insensitive dihydro-2, the dipicolimic acid 2 synthase.

13. comprise the carrier or the plasmid of each nucleic acid in the claim 1 to 12.

14. comprise in the claim 1 to 12 each the nucleic acid or the host cell of the carrier of claim 13.

15. the host cell of claim 14, it comprises edaphic bacillus.

16. the host cell of claim 15, it comprises the edaphic bacillus that virulence is arranged basically.

17. the host cell of claim 15 or 16, wherein said edaphic bacillus comprises Agrobacterium tumefaciens.

18. the host cell of claim 17, wherein said host cell comprise Agrobacterium tumefaciens A281 or by its deutero-cell.

19. comprise the vegetable cell of each nucleic acid in the claim 1 to 12.

20. derive from plant or its part of the vegetable cell of claim 19.

21. the tuberous plant of claim 20 or its part.

22. the plant of claim 21 or its part, it is selected from potato or cassava plant.

23. the method for transformed plant cells comprises each nucleic acid in the claim 1 to 12 is offered vegetable cell.

24. the method for claim 23 further is included in to derive from and cultivates described cell under the essentially no selective pressure condition of selecting substratum.

25. the method for claim 23 or 24 further comprises the functional at least part that whether has each nucleic acid in the claim 1 to 12 in the described cell filial generation of test at least a portion.

26. each method in the claim 23 to 25 further comprises in the described cell filial generation of test at least a portion whether have non-required vector nucleic acid.

27. by the obtainable vegetable cell of each method in the claim 23 to 26.

28. derive from plant or its part of the vegetable cell of claim 27.

29. the tuberous plant of claim 28 or its part.

30. the plant of claim 29 or its part, it is selected from potato or cassava plant.

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